Medicament containing a tissue inhibitor of metalloproteinases-2 (TIMP-2) as an osteoanabolically active substance

ABSTRACT

The invention relates to tissue inhibitor of metalloproteinase-2 (TIMP-2) as an osteoanabolically active peptide for use as a medicament for treating bone defects, bone diseases and for improving bone regeneration.

This application is a 371 of PCT/EP01/04891, filed Apr. 4, 2001.

This is a nationalization of PCT/EP01/04891, filed Apr. 4, 2001 andpublished in German.

The invention relates to a medicament and a diagnostic agent containingtissue inhibitor of metalloproteinases-2 (TIMP-2) as anosteoanabolically active peptide preparation, and the use thereof.

TIMP-2 belongs to the family of “tissue inhibitors ofmetalloproteinases”, of which four different peptides (TIMP-1 to TIMP-4)having different biological functions have been known to date.

The primary structure of TIMP-2 could be elucidated for variousorganisms, such as humans (Liotta et al., 1991), rats (Roswit et al.,1992), and mice (Kishi et al., 1991). The peptide contains a total of 12cysteines which are linked to one another through 6 cystine bridges.

The biological functions of TIMP-2 include, inter alia, the inhibitionof active matrix metalloproteinases (MMPs). The term MMPs refers to agroup of zinc-dependent endoproteinases which are capable of degradingthe extracellular matrix. The extracellular matrix consists of a complexstructure and is constituted, inter alia, of collagen, proteoglycans,glycoproteins and glycosaminoglycans. Degradation of the extracellularmatrix is essential to many biological processes, such asembryo-genesis, morphogenesis and tissue absorption and remodelling.However, uncontrolled activity of the MMPs can result in a considerabledestruction of tissues, such as in rheumatic arthritis (Okada et al.,1986).

Further biological functions of TIMP-2 are related to the inhibition ofthe matrix metalloproteinases, including, inter alia, reduction of thegrowth of tumor cells (Gomez et al., 1997), and inhibition ofangiogenesis (Valente et al., 1998).

In addition to these functions, intrinsic biological activities ofTIMP-2 have also been described. These include an increased formation ofred blood cells (Stetler-Stevenson et al., 1992), and a mitogenic effecton different cell lines (Hayakawa et al., 1994). Further, bothinhibition of bone absorption in whole bone cultures (Hill et al., 1993)and stimulation of bone absorption by osteoclasts (Shibutani et al.,1999) could be shown in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1D show different chromatographic steps for the purification ofosteoproliferative TIMP-2.

FIGS. 2A–2B show that proliferative effect of recombinant human TIMP-2on primary fetal rat osteoblasts. B) Influence of recombinant humanTIMP-2 on the average cell diameter in the mouse osteoblastic cell lineMC3T3-E1.

Surprisingly, TIMP-2 having the amino acid sequence

Z¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGP(residues 1–56(Z¹-SEQ ID NO: 3-Z²) of SEQ NO:1)X1KDTEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTL (residues58–110 of SEQ ID NO: 1)X2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEK(residues 112–155 of SEQ ID NO:1)X3INGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP (residues 157–194 of SEQ IDNO: 1)-Z²

wherein X1 represents the amino acid E or D; X2 represents the aminoacid T or I; X3 represents the amino acid N or S; Z¹ represents —H, asubstituted group or an arbitrary peptide having up to ten amino acids;Z² represents —OH, NH₂, a substituted —NH₂ or an arbitrary peptidehaving up to ten amino acids;and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives, are capable of causing stimulation of theosteoblasts responsible for osteogenesis. TIMP-2 has a proliferative andprotective effect on fetal osteoblasts.

Therefore, according to the invention, a medicament is claimedcontaining TIMP-2 having the amino acid sequence

(Z¹-SEQ ID NO: 3-Z²) Z¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPX1KDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3INGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIED P-Z²

wherein X1 represents the amino acid E or D; X2 represents the aminoacid T or I; X3 represents the amino acid N or S; Z¹ represents —H, asubstituted group or an arbitrary peptide having up to ten amino acids;Z² represents —OH, NH₂, a substituted —NH₂ or an arbitrary peptidehaving up to ten amino acids;and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives.

According to the invention, there are preferably employed human TMP-2(SEQ ID NO.1) and rat TIMP-2 (SEQ ID NO.2):

CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPEKD (SEQ. ID.NO 1) IEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKAEGDGKMHITLCDFIVPWDTLSTTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDPCSCSPVHPQQAFCNADVVIPAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPDKD (SEQ. ID. NO2) IEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLSITQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKSINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP

The invention also relates to the use of TIMP-2 having the amino acidsequence

Z¹CSCSRVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPX1KDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3INGHQAKFFACIKRSDGSCAWYRGAAPP KQEFLDIED P-Z²

wherein X1 represents the amino acid E or D; X2 represents the aminoacid T or I; X3 represents the amino acid N or S; Z¹ represents —H, asubstituted group or an arbitrary peptide having up to ten amino acids;Z² represents —OH, NH₂, a substituted —NH₂ or an arbitrary peptidehaving up to ten amino acids;and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives for preparing an osteoanabolic medicament.

TIMP-2 derivatives which can be used according to the inventionpreferably have at least 90% sequence identity with the native sequenceof TIMP-2 or the mentioned modifications.

It may be advantageous to employ the TIMP-2 to be used according to theinvention in combination with other growth factors and/or medicamentsand in combination with medical aids, for example, in grafts.

The administration of TIMP-2 is effected, in particular, as aformulation in the form of injections, ointments, sustained releasecapsules and similar galenic formulations.

The invention also relates to the use of low-molecular weight substancesand active compounds which have effects on bone cells comparable tothose of TIMP-2 for preparing a medicament for treating diseases of thebone system, such as fractures, osteopenia and osteoporosis.

Further, the invention also relates to the use of a receptor for TIMP-2on bone cells in connection with its related agonists and antagonistsfor preparing a medicament for diseases of the bone system, such asfractures, osteopenia and osteoporosis.

The TIMP-2 to be used according to the invention is employed, inparticular, for preparing an osteoanabolic medicament for treating bonedefects and for improving bone regeneration, especially following bonefractures or surgical intervention, and for treating degenerative andmetabolic bone diseases, such as osteoporosis, osteopenia andosteomalacia, and inflammatory bone diseases, such as ostitis andosteomyelitis.

The invention also relates to a diagnostic agent for functionaldisorders of the bone, containing TIMP-2 having the amino acid sequence

(Z¹-SEQ ID NO: 3-Z²)Z¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPX1KDIEFIYTAPSSAVCGSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3INGHQAKFFACIKRSDGSCAWYRGAAPP KQEFLDIEDP-Z²

wherein X1 represents the amino acid E or D; X2 represents the aminoacid T or I; X3 represents the amino acid N or S; Z¹ represents —H, asubstituted group or an arbitrary peptide having up to ten amino acids;Z² represents —OH, NH₂, a substituted —NH₂ or an arbitrary peptidehaving up to ten amino acids;and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives.

The basis of the identification of TIMP-2 as an osteoanabolic peptidewas the observation that there is a different ability of regeneration ofbone defects depending on the age of an organism. While adult organismsare not normally capable of regenerating major bone defects by theformation of new bone tissue, a complete bony regeneration of comparabledefects takes place in younger organisms.

Similar observations were also made in vitro in the examination ofosteoblasts: Osteoblasts isolated from fetal organisms grow without anyproblems in cultures and begin to mineralize after a few days. Incontrast, osteoblasts from immature and adult animals are not capable ofproliferating under standard culture conditions and eventually die. Ifthese cells, which are not viable alone, are supplemented with thesupernatant of fetal osteoblasts, they proliferate and mineralize in away similar to the fetal cells.

A clearly regenerative capability of the cell culture supernatant offetal osteoblasts could also be shown in vivo. Thus, a skull defectwhich cannot heal with the formation of bone tissue without externalstimulation was surgically caused to rats. However, if a peptide extractfrom the cell culture supernatant of fetal primary osteoblasts isadministered into these defects, then a complete regeneration of thedefect takes place with the formation of new bone tissue.

In order to isolate the substance which is responsible for the effectsdescribed above, a peptide extraction from the supernatant of fetalosteoblasts was performed, and the peptides obtained were subsequentlyexamined in vitro for their osteoproliferative capabilities (see Example3). The peptide according to the invention could be purified therebyusing preparative, semipreparative and analytical RP chromatography dueto its biological activity.

The biochemical characterization of the purified peptide according tothe invention was effected by mass spectrometry and N-terminalsequencing. Determination of the molecular mass by electrospray massspectrometry yielded a molecular mass of 21,715 Da. The N-terminalsequence analysis by Edman degradation resulted in the followingsequence:

XSXSPVHPQQAFXNADVVIRAKAV (SEQ ID NO:4)

The substance according to the invention can be employed to induceenhanced growth and improved regeneration of bone tissue. This may beconsidered, in particular, after bone fractures or surgicalintervention, but also in systemic bone diseases.

The invention will be illustrated in more detail by the followingExamples:

EXAMPLE 1

Isolation of TIMP-2 from Cell Culture Supernatant

Cell culture supernatants were obtained from confluent cultures ofprimary fetal osteoblasts. Thus, the osteoblasts were first isolatedfrom the skull caps of rat fetuses by sequential digestion withcollagenase and trypsin, followed by cultivation in Eagle's minimalessential medium (MEM) with penicillin/streptomycin and 10% fetal calfserum (FCS). After confluency was reached, the cells were keptalternately with 10% FCS for 24 hours and serum-free for 24 hours. Theserum-free supernatants were collected and used for further peptideisolation.

The isolation of TIMP-2 was effected by various subsequent RP-HPLC stepswhich served for the fractioning of the whole peptide extract. Aliquotsof the fractions were tested in a bioassay. The fractions were stored at−20° C.

The different chromatographic steps for the purification ofosteoproliferative TIMP-2 are shown in FIGS. 1 A–D.

1. Preparative Chromatography

The serum-free cell culture supernatants were adjusted with HCI to a pHof 2.5 and by the addition of acetonitrile to 5% acetonitrile.Subsequently, they were filtered first over a glass fiber filter (GF6,Schleicher & Schuell) and then over a membrane filter (OE G7, 4.43 μm,Schleicher & Schuell). The material thus obtained was charged onto a RPcolumn in amounts of from 1 to 2 l with a flow rate of from 40 to 50ml/min.

Chromatographic conditions:

Column: YMC Gel Basic (15–30 μm, 47 × 300 mm) Flow: 40 ml/min Fractions:50 ml Buffer A: 10 mM HCl Buffer B: 80% (w/v) acetonitrile, 10 mM HClGradient: 5 to 75% B in 47.5 min 75 to 100% B in 5 min.

Subsequently, aliquots of the fractions obtained were tested in abioassay.

2. Semipreparative Separation

Fractions 37–40, which were bioactive in the assay, were separated bymeans of a further RP step.

Chromatographic conditions:

Column: Biotek RP Silica C4 (100 A, 5 μm, 20 × 125 mm) Flow: 5 ml/minFraction: 5 ml Buffer A: 0.1% TFA Buffer B: 80% (w/v) acetonitrile, 0.1%TFA Gradient: 5 to 40% B in 10 min 40 to 100% B in 60 min.

Aliquots of the fractions obtained were again tested in a bioassay.

3. Analytical Chromatography

The subsequent fractioning of the bioactive fractions 22 and 23 waseffected by means of another RP chromatography.

Chromatographic conditions:

Column: YMC C18 (120 A, 5 μm, 4.6 × 250 mm) Flow: 0.6 ml/min Fraction:0.6 ml Buffer A: 0.1% TFA Buffer B: 80% (w/v) acetonitrile, 0.1% TFAGradient: 5 to 30% B in 4 min 30 to 55% B in 50 min 55 to 100% B in 5min.

Aliquots of the fractions obtained were again tested in a bioassay.

4. Analytical Chromatography

The bioactive fractions 27–29 were again separated by means of RPchromatography.

Chromatographic conditions:

Column: Phenomenex C5 (4.6 × 250 mm) Flow: 0.6 ml/min Fraction: 0.6 mlBuffer A: 0.1% TFA Buffer B: 80% (w/v) acetonitrile, 0.1% TFA Gradient:5 to 38% B in 4 min 38 to 58% B in 60 min 58 to 100% B in 5 min.

In the subsequent biotest, the peptide according to the invention couldbe obtained in pure form in fraction 24.

EXAMPLE 2

Biochemical Analysis of TIMP-2

1. Mass Determination

The mass determination of the native purified peptide was performed bymeans of an ESI mass spectrometer. A molecular mass of 27,715 Da wasobtained. This is in very good agreement with the theoretical mass ofthe oxidized form of TIMP-2 in which all the cysteines are bridged bycystine bridges.

2. Sequence Determination

The purified native peptide was analyzed by Edman degradation on an ABI494 sequencer using the standard program. The first 24 degradation stepsyielded the following N-terminal sequence:

-   XSXSPVHPQQAFXNADVVIRAKAV (SEQ ID NO:4)

X are cysteine residues which cannot be detected by sequencing. Takinginto account the non-detectable cysteines, the sequence obtained isexactly identical with the known sequence of TIMP-2. Minor sequenceswere not obtained.

3. Data Base Search

In a subsequent alignment in the data base SwissProt, the sequence wasunambiguously identified as the beginning of TIMP-2. The theoreticalmass of the protein of 27,713 Da is also in agreement with the measuredmass of 27,715 Da. Thus, the purified peptide could be unambiguouslyidentified as TIMP-2.

EXAMPLE 3

Determination of the Biological Peptide of TIMP-2

The isolation of TIMP-2 was effected by means of its biologicalactivity, the proliferation of fetal rat osteoblasts being measured.

Thus, aliquots of the peptide fractions obtained were lyophilized andresuspended in serum-free medium. Subsequently, their proliferativeeffect on fetal osteoblasts was tested. Subsequently, fractionsexhibiting a proliferative effect were subjected to anotherchromatographic separation. The different chromatographic steps for thepurification of osteoproliferative TIMP-2 are shown in FIGS. 1 A–D.

For proliferation measurement, the primary osteoblasts were trypsinizedand plated in 96 well plates in a serum-free medium in a cell density of5000 cells per well. Subsequently, the fractions to be tested wereadded. After an incubation time of 48 h or 72 h, the proliferation wastested by means of two different methods. As positive controls,different concentrations of fetal calf serum and TGFβ1 were used. Cellshaving obtained no stimulation served as a negative control.

On the one hand, proliferation measurement was effected by means of theWst-1 substrate. This substrate is converted by mitochondrial enzymes inmetabolically active cells to a colored product, the color intensityformed being proportional to the number of cells. The color intensity isdetermined at a wavelength of 405 nm and a reference wavelength of above600 nm in an ELISA reader.

On the other hand, the proliferation is measured by direct measurementof the cell number in a Coulter counter (CASY).

Both methods showed a dose-dependent growth-promoting effect ofrecombinant human TIMP-2 on osteoblasts (FIG. 2A). A significantosteoproliferative effect of TIMP-2 can be detected in these in-vitroexperiments from concentrations of 100 ng/ml. In addition, human TIMP-2causes a decrease of the average cell diameter in the mouse osteoblasticcell line MC3T3-E1 (FIG. 2B).

Significant effects of TIMP-2 can be detected in this in-vitroexperiment from concentrations of 100 ng/ml. Further, a protective andproliferative effect on the mouse osteoblastic cell line MC3T3-E1 couldbe shown by microscopy. Since all these cells are typical bone cells,TIMP-2 can be considered an osteoanabolic factor.

1. A method of stimulating osteoblasts after bone fracture or surgicalintervention comprising administering an effective amount of astimulating agent in combination with a graft to a patient in needthereof, wherein the stimulating agent is a) tissue inhibitor ofmetalloproteinases-2 (TIMP-2) having the amino acid sequenceZ¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEI KQIKMFKGP(residues1–56 of SEQ ID NO:1) X1KDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTL (residues 58–110 of SEQ ID NO:1)X2TTQKKSLNHRYQMGCECKITRCPMIPCYISSRDECLWMDWVTEK (residues 112–155 of SEQID NO:1) X3INGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP (residues 157–194 ofSEQ ID NO:1)-Z²

wherein X1 is the amino acid E or D, X2 is the amino acid T or I, X3 isthe amino acid N or S, Z¹ is hydrogen or a substituent group or anarbitrary peptide having up to ten amino acids, and Z² is —OH, —NH₂,substituted —NH₂ or an arbitrary peptide having up to ten amino acids,or b) the TIMP-2 of (a) modified by amidation, acylation, sulfation,phosphorylation, glycosylation, attachment of polyethylene-glycolgroups, or a combination thereof, wherein the modified TIMP-2 hasosteoblast stimulating activity after a bone fracture or surgicalintervention, thereby stimulating osteoblasts.
 2. The method of claim 1,wherein the stimulating agent is in a galenic formulation.
 3. The methodof claim 2, wherein the galenic formulation is an injection preparation,an ointment, or a sustained release capsule.